Alteration of the antigenicity of human lymphocytes by plant lectins and short-term tissue culture.

Human lymphocytes studied after being placed in culture for 1–6 wk progressively lost stimulating ability, i.e., lymphocyte defined antigens, when tested in one way mixed lymphocyte culture (MLC) but retained several other identifiable membrane components as well as the capacity to respond to mitogenic stimuli. Lymphocytes placed in culture with motogenic doses of PHA and Con-A after 1 and 2 wk strongly stimulated autologous responding fresh lymphocytes, but the MLC response of allogeneic fresh lymphocytes to stimulating lectin treated cells was even lower than the response to stimulating allogeneic cultured lymphocytes. The HL-A antigens on lectin treated cells or on lymphocytes through 6 wk in culture were clearly identifiable. Assays for T cell rosettes and B cell surface immunoglobulin showed both cell types to be present in numbers equal to fresh lymphocytes for up to 5 wk after culturing. However, the Fc receptor site on B cells was lost from cultured lymphocytes at the same time that MLC stimulation was lost. It is concluded that plant lectins can unmask new mitogenic sites on the cell surface as well as mask or delete existing sites, and that culturing lymphocytes for 1–6 wk will produce somewhat similar modulations. Modulation of surface membrane components by tissue culture or lectins may, therefore, have a profound effect in altering transplantation immunogenicity.     

Cross-reactivity between human and murine lymphocyte antigens: III. Reactivity of H-2 allo- and xenoantisera with human lymphoid cells

H-2 alloantisera and antimouse lymphocyte xenoantisera react with 14%–100% of human lymphocytes from a panel of at least 80 unrelated people. Population and family studies did not reveal HL-A specificity of such lymphocytotoxic antibodies but indicated that the antibodies are directed against polymorphic antigenic determinants inherited in association with HL-A antigens. H-2 allo- and xenoantisera absorbed with human lymphoid cells and a panel of platelets bearing all the known HL-A specificities were still cytolytic when tested against murine lymphocytes, suggesting that only a small proportion of the heterogeneous population of H-2 antibodies react with human lymphocytes. On the other hand, HL-A alloantisera could be absorbed by lymphocytes from certain murine strains. These results suggest that the crossreactivity between human and murine lymphocytes is caused by antigens common to several HL-A (or H-2) types or by antigens linked to HL-A but not identical with them.     

Membrane-associated antigens of human malignant melanoma IV: Changes in expression of antigens on cultured melanoma cells

Summary Established melanoma cell lines were cultured for one passage (approximately 1 week) in different lots of fetal calf and new born calf sera and then tested against a panel of previously positively reacting sera from melanoma patients and polyspecific HL-A alloantisera. Using indirect immunofluorescence the cells showed varying degrees of reactivity ranging from positive to negative reactions depending on the supplementing serum in the culture medium. When standardized culture conditions were used and the cells were tested by immune adherence at several weeks intervals against panels of sera from melanoma patients, from tumor patients other than melanoma, from pregnant women, and from normal donors, most of the sera reacted identical, but some sera not only had changed quantitatively but also qualitatively from a negative to a positive reaction and vice versa indicating a shift in the spectrum of expressed antigens. When single cell clones from a cell line were isolated and tested against a panel of antisera, striking differences in reactivity were observed suggesting that the shift in the spectrum of expressed antigens was due to the outgrowth of dominating subclones with antigen patterns different from the previously dominating subclones. This conclusion was further supported by experiments in which a weakly positive reacting serum was employed to separate a cell line into positively and negatively reacting sublines. Unit gravity sedimentation and density gradient sedimentation were used in order to separate rosetted from non-rosetted tumor cells which had been prepared by immune adherence. It is concluded that cultured cell lines are in a dynamic state and that differentiation is one of the major mechanisms accounting for a change in antigen expression.     

Characterization of a serum inhibitor of MLC reactions. III. Specificity: HL-A-related antibodies.

A mixed leukocyte culture (MLC)-inhibitory serum from a healthy multipara, JH, has been characterized with regard to the specificity of its inhibitory antibody. When added directly to MLC, JH serum will inhibit most combinations. However, when lymphocytes intended as responder or stimulator cells in MLC were preincubated with this serum, the specificity narrowed considerably. Four groups of lymphocyte donors were recognized, depending upon whether their lymphocytes were inhibited as responders, as stimulators, as both, or as neither. Absorptions of inhibitory activity, followed by assay of the absorbed sera in pretested MLC combinations, yielded reliable data for determining which donors' cells shared pertinent antigens. An association of MLC inhibition by JH serum with the HL-A types of the involved lymphocytes was observed and these relationships are summarized in Table 4. The three HL-A specificities identified, W19, W29, and 12, correspond with the HL-A typing of the husband of the serum donor. Various cell types absorbed relevant inhibitory activity (against responder and stimulator functions) in the following order of efficiency: LCL cells, B lymphocytes, T lymphocytes, fibroblasts, and erythrocytes. When the above three HL-A specificities were removed by absorption, the serum was no longer inhibitory in any combinations. Whether the inhibitory activity of JH serum is directly related to anti-HL-A antibodies or to antibodies against closely related MLR determinants will depend to a large extent upon the degree of linkage disequilibria found between W19, W29, and 12 antigens and the MLR locus.     

The human mixed-lymphocyte culture (MLC) interaction after in vivo allo-immunization. II. HL-A specificity of early proliferative response

Human volunteers were immunized with a single intradermal dose of allogeneic buffycoat cells. When donor and recipient differed for four HL-A antigens belonging to the first and second series, an increased MLC proliferation early in the cultures was observed when responding lymphocytes from the immunized individual were confronted with stimulating cells from his donor. These findings were not observed when the incompatibility between donor and recipient involved only one second series HL-A antigen. The specificity of the altered response was studied by confronting responding lymphocytes from the immunized individual with different third-party stimulating cells. Most of these combinations revealed an early increased proliferation compared to control combinations with responding lymphocytes from nonsensitized individuals. However, the early increased responsiveness was significantly more pronounced in combinations where the stimulating cells shared two or more “private” HL-A determinants with the immunizing donor. It is concluded that a major part of the early increased responsiveness is due to proliferation of lymphocytes with receptors for HL-A (SD) determinants.     

Human platelets as a source of HL-A antigens : a study of various solubilization techniques.

The efficiency of various methods of solubilizing HL-A platelet antigens was investigated. The yield of soluble material was compared with that obtained from lymphocytes in culture in order to judge the quality of platelets as a source of HL-A antigens. The conclusion was reached that platelets, easily obtainable, can be considered as a good source of HL-A antigens.     

The effect of heating and drugs on HL-A antigens and their reactions with cytotoxins]

In some instances the heating of lymphocytes for 2 to 3 minutes at 56 degrees C enhances HL-A antigens or makes it possible to detect these antigens by a twostage microlymphocytotoxic test on preheated lymphocytes only. Similar phenomenons are observed in some tannin (1: 40 000) or 0.1% phenol-treated lymphocytes and after the addition of these solutions during the first stage of the lymphocytotoxic test. Prolonged heating at 56 degrees C leads to nonspecific polyreactivity of lymphocytes, giving false results with all sera. For similar reasons also higher concentrations of tannin and phenol solutions were found dissatisfactory for the pretreatment of lymphocytes. The pretreatment of lymphocytes with 36-0.5% formaline induces full inhibition of specific cytotoxic reactivity of HL-A antigens and their absorption ability with regards to respective HL-A sera. The addition of formaline at 0.06-0.3% concentration during the first and second stages of the test and at the end of the second stage (or simultaneously with eosine) gives also negative results of the cytotoxic test owing to the inhibitory effect of formaline on rabbit complement and HL-A antigens.     

Replication and persistence of measles virus in defined subpopulations of human leukocytes.

Replication of Edmonston strain measles virus was studied in several human lymphoblast lines, as well as in defined subpopulations of circulating human leukocytes. It was found that measles virus can productively infect T cells, B cells, and monocytes from human blood. These conclusions were derived from infectious center studies on segregated cell populations, as well as from ultrastructural analyses on cells labeled with specific markers. In contrast, mature polymorphonuclear cells failed to synthesize measles virus nucleocapsids even after infection at a relatively high multiplicity of infection. Measles virus replicated more efficiently in lymphocytes stimulated with mitogens than in unstimulated cells. However, both phytohemagglutinin and pokeweed mitogen had a negligible stimulatory effect on viral synthesis in purified populations of monocytes. In all instances the efficiency of measles virus replication by monocytes was appreciably less than that of mitogenically stimulated lymphocytes or of continuously culture lymphoblasts. Under standard conditions of infection, all of the surveyed lymphoblast lines produced equivalent amounts of measles virus regardless of the major histocompatibility (HL-A) haplotype. Hence, no evidence was found that the HL-A3,7 haplotype conferred either an advantage or disadvantage with respect to measles virus synthesis in an immunologically neutral environment. A persistent infection with measles virus could be established in both T and B lymphoblasts. The release of infectious virus from such persistently infected cells was stable over a period of several weeks and was approximately 100-fold less than peak viral titers obtained in each respective line after acute infection.     

Recognition of HLA-A2 mutant and variant target cells by an HLA-A2 allospecific human cytotoxic T lymphocyte line

HLA-A2 specific human cytotoxic T lymphocytes (CTL) cell lines have been developed using T cell growth factor and coculture of peripheral blood lymphocytes with selected allogeneic target cell lines. The CTL-8 line showed specificity for human leukocyte antigens (HLA)-A2 bearing target cells after 5 weeks in culture when tested against a panel of 14 lymphoblastoid cell lines in a 51Chromium (51Cr) release assay. Purified anti-human leukocyte antigens (HLA) monoclonal antibodies W6/32 and PA2.1 inhibited cytolysis by 85% and 60%, respectively. The CTL-8 line lysed non-HLA-A2 target cells in the presence of lectins concanavalin A (Con A) or phytohemagglutinin-P lectin (PHA-P) indicating the specificity of cytolysis was not due to nonspecific resistance of target cells to the CTL-lytic mechanism. The T5-1 HLA-A2 mutant cell series were tested as targets for the CTL-8 line. Cell clones 8.18.1, 8.21.1 and 8.6.1, which express altered HLA-A2 molecules as determined by their decreased reactivity with allospecific monoclonal antibodies, were lysed by the CTL-8 line as efficiently as the T5-1 wild type. These cell lines also acted as efficient cold target competitors for a normal HLA-A2 target cell. The 8.14.1 cell clone expressed a lower amount of HLA-A2 alloantigen and showed a corresponding decreased reactivity with CTL-8 in direct cytolytic and cold target competitive inhibition assays. In contrast, the M7 and DK1 HLA-A2 variant cell lines, which express normal HLA-A2 serological determinants, were inefficiently lysed by CTL-8 and did not act as competitive inhibitors of normal HLA-A2 target cells. These results support the concept that the alloantigenic determinant(s) recognized by T cells and antibodies occur at separate regions on the HLA-A2 molecule.     

HL-A genotype of patients with acute lymphoblastic leukaemia

Summary 10 patients with acute lymphoblastic leukaemia were tissue-typed for 21 HL-A specificities. Of these, genotypes of 9 pateints were determined by family analyses. Haplotype HL-A1,8 occurred in 5 out of 18 instances. On phenotype basis, a slight increase was observed in the incidence of antigens HL-A1 and HL-A8. No loss of HL-A specificities could be detected on lymphocytes through family analyses.     

Interleukin-2- and mitogen-activated NK-like killer cells from highly purified human peripheral blood T cell (CD3+ N901-) cultures

In this study, highly purified (HP) CD3-positive N901-negative T lymphocytes could be induced to become natural killer (NK)-like in culture in the presence of recombinant interleukin-2 (rIL-2) and phytohemagglutinin (PHA). Thus, purified CD3+ N901- T cells from fresh human peripheral blood were obtained by negative selection using an indirect panning technique. To ensure that T lymphocyte fractions were completely devoid of any detectable NK cells, two additional purification procedures were employed: incubation of post-pan T cells with the NK-cytotoxic lysomotropic agent L-leucinemethylester, and complement-mediated lysis using the NK cell specific NKH1a monoclonal antibody. Purity of CD3+ N901- cells could be confirmed by surface marker analysis, whereby two NK-associated antigens, N901 and H-25, were undetectable, while 94 +/- 1% of cells expressed the CD3 (Leu-4) antigen. On functional analysis, fresh HP CD3+ N901- cells exhibited no cytotoxic activity against the standard NK target K562. When HP NK-depleted T lymphocytes were cultured for 7 days in the presence of rIL-2 (100 U/ml), neither surface antigen expression nor cytotoxic activity against K562 changed significantly. However, significant cytotoxicity against K562 [18 +/- 5% specific lysis at 25:1 effector:target (E/T) ratio] could be induced when HP CD3+ N901- cells were grown for 7 days in the presence of rIL-2 and PHA (0.5% v/v). Concomitantly, antigens N901 and H-25 were found to be coexpressed on a minor proportion (22 +/- 16 and 22 +/- 6%, respectively) of CD3+ (88 +/- 2% on day 7) cells. Four-week long-term culture of HP NK-depleted T cells in the presence of rIL-2 and PHA yielded a continuous increase in cytotoxicity against K562 cells (0 up to 46% specific lysis at 25:1 E/T ratio). Of particular interest was the emergence of cytotoxicity against the NK-resistant Daudi cell target (15 +/- 8% specific lysis at 25:1 E/T ratio on day 21). Expression of antigens N901 and H-25 as well as CD3 remained essentially unchanged in long-term culture. In sorting experiments, the H-25+ cell fraction was significantly enriched for cytotoxicity against K562, when compared to both H-25- and unseparated cell fractions. In summary, our results suggest that a proportion of HP CD3+ N901- T lymphocytes may give rise to cells that exhibit NK-like functional and phenotypic properties.     

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal mntibodies in cryostat sections of biopsies

Summary Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.     

Genetic relationships between the HL-A system, the blood group systems (Rh, ABO) and the immune reactivity]

The authors examined in immunized healthy persons the correlations between the ability of immune response, the value of their different immunological parameters, and the HL-A blood-group antigens by computer analysis. Immune reactivity showed mosaic-like correlation against the HL-A system. The most definite negative correlation was noticed between the HL-A 3 and 7 antigens and the cytotoxic activity of lymphocytes. Remarkable and definite correlation was found between the Rh system and immune reactivity. The level of the natural antibodies, the immunoglobulins and the functions of lymphocytes were generally decreased in males in comparison to females.     

Inhibition of T-lymphocyte rosetting by HL-A alloantisera and complement.

The relationship between HL-A antigens and rosetting of sheep red blood cells (SRBC) with peripheral human lymphocytes has been investigated by incubating them with HL-A antibodies. Although sensitizing the lymphocytes with HL-A alloantisera had no effect on their ability to form rosettes with SRBC, further sensitization with C6 deficient rabbit serum as a source of early complement components inhibited the formation of rosettes with SRBC. The involvement of HL-A alloantibodies in the inhibition of rosette formation was shown first by correlating the HL-A phenotype of the lymphocytes and the HL-A specificity of the alloantisera and, second, by specifically absorbing the HL-A alloantibodies from the alloantisera. Complement was needed to inhibit rosette formation since this effect was lost when rabbit serum was treated to inactivate complement. The participation of complement's classical pathway in rosette inhibition was shown by chelating the Ca2+ ions by EGTA treatment of the C6 deficient rabbit serum. Perhaps, binding of HL-A antibodies and early complement components to the lymphocyte surface disturbs the distribution of the receptors or affects the charge of the cell membrane, thus inhibiting the rosette formation with SRBC.     

Immune function of the blood-brain barrier: incomplete presentation of protein (auto-)antigens by rat brain microvascular endothelium in vitro.

The endothelial blood-brain barrier (BBB) has a critical role in controlling lymphocyte traffic into the central nervous system (CNS), both in physiological immunosurveillance, and in its pathological aberrations. The intercellular signals that possibly could induce lymphocytes to cross the BBB include immunogenic presentation of protein (auto-)antigens by BBB endothelia to circulating T lymphocytes. This concept has raised much, though controversial, attention. We approached this problem by analyzing in vitro immunospecific interactions between clonal rat T lymphocyte lines with syngeneic, stringently purified endothelial monolayer cultures from adult brain micro-vessels. The rat brain endothelia (RBE) were established from rat brain capillaries using double collagenase digestion, density gradient fractionation and selective cytolysis of contaminating pericytes by anti-Thy 1.1 antibodies and complement. Incubation with interferon-gamma in most of the brain-derived endothelial cells induced Ia-antigens in the cytoplasm and on the cell surface in some of the cells. Before the treatment, the cells were completely Ia-negative. Pericytes were unresponsive to IFN-gamma treatment. When confronted with syngeneic T cell lines specific for protein (auto-)antigens (e.g., ovalbumin and myelin basic protein, MBP), RBE were completely unable to induce antigen-specific proliferation of syngeneic T lymphocytes irrespective of pretreatment with IFN-gamma and of cell density. RBE were inert towards the T cells, and did not suppress T cell activation induced by other "professional" antigen presenting cells (APC) such as thymus-derived dendritic cells or macrophages. IFN-gamma-treated RBE were, however, susceptible to immunospecific T cell killing. They were lysed by MBP-specific T cells in the presence of the specific antigen or Con A. Antigen dependent lysis was restricted by the appropriate (MHC) class II product. We conclude that the interaction of brain endothelial cells with encephalitogenic T lymphocytes may involve recognition of antigen in the molecular context of relevant MHC products, but that this interaction per se is insufficient to initiate the full T cell activation program.     

Changes in HL-A antigen profiles on SV40-transformed human fibroblasts

The histocompatibility antigen profile of human fibroblasts transformed with SV40 virus has been investigated by determining their ability to specifically absorb HL-A alloantisera. Four out of four human adult skin fibroblast lines acquired, after SV40 transformation, the ability to absorb the HL-A alloantiserum VICTOR directed against the specificities HL-A5, W5. On the contrary, none of six embryonic fibroblast lines showed any qualitative or quantitative change of their HL-A antigenic profile. Similarly, murine, monkey and hamster cells could not absorb the activity of the HL-A alloantiserum VICTOR after SV40 transformation. It is suggested that the ‘new HL-A antigen’ specificity on human fibroblasts after SV40 transformation may be due to either a cross-reaction between HL-A specificities and antigenic structures present on glycoprotein(s) coded by the viral genome and expressed on the cell or to a change in the regulating mechanism of the expression of histocompatibility antigens on the cell surface, should the genetic information for all the HL-A specificities be present in the cell genome.     

Studies on changes in lymphocyte HL-A antigens during the course of malignant diseases]

The presence of HL-A antigens 1, 2, 5, 7, 8 and 12 on the lymphocytes of 26 patients with blood diseases and malignant tumours was examined by means of the two-step microcytotoxicity test. The studies carried out several times in the course of the disease and with 4 to 5 sera of the same specificity. Two types of the serological modifications were found: 1. Transient loss of HL-A antigens in 6 patients, 2. Evidence of the polyreactivity of lymphocytes in 3 patients. The polyreactivity was later changed to the loss of HL-A antigens. In one patient, the destruction of lymphocytes in the course of the testing was proved. The importance of the results is discussed. The additional serological, morphological and clinical investigations seems to be necessary.     

The restoration of proliferation and differentiation of peripheral blood mononuclear non-adherent cells into immunoglobulin-secreting cells by autologous synovial adherent cells from patients with rheumatoid arthritis

The ability of enzyme-dissociated synovial adherent cells (SAC) obtained from patients with active rheumatoid arthritis to restore the proliferation and differention of peripheral blood mononuclear non-adherent cells (NAC) into immunoglobulin-secreting cells (ISC) was investigated. Autologous combinations of cells were used in this study to eliminate allogeneic reactions. Peripheral blood NAC, prepared by glass adherence and leucine methylester treatment to remove monocytes, almost completely lost their capacity to proliferate and differentiate into ISC in response to pokeweed mitogen. The response of NAC was restored by adding 12.5% of 'fresh SAC', which was obtained by glass-adherence after an overnight culture of non-rosette forming, enzyme-dissociated rheumatoid synovial cells. Although the response was also restorable by adding more than 25% fresh SAC, this was less satisfactory than adding 12.5% SAC. 'Old SAC', obtained by glass-adherence after 7 days culture of enzyme-dissociated synovial cells, did not restore the response of NAC. Immunohistochemical studies showed that 55% of fresh SAC and 3% of old SAC expressed HLA-DR antigens. When 100 units/ml of interferon gamma was present, 25% of old SAC remained HLA-DR-positive and some of these cells retained a dendritic morphology after 7 days culture. The results indicate that rheumatoid synovia contain macrophage-like cells that can effectively support the ultimate differentiation of lymphocytes to ISC.     

Autoimmunity to glial antigens in vitro.

Lymph nodes cells and spleen cells in a 50:50 mixture from Fischer 344 rats were cultured on syngeneic glial cells and fibroblasts. In the glia cultures, but not in the fibroblast cultures, the lymphocytes were stimulated to a vivid blast transformation and mitotic activity with a peak after 7 days, after which they reverted to small- and medium-sized lymphocytes. The stimulated lymphoid cells were not cytotoxic to glial cells when tested in a microcytotoxicity assay. A fraction of the lymphoblasts and their progeny (approximately 4%) took a positive intracytoplasmic stain for γ-globulin immunoglobulin G after direct immunofluorescence. Efforts to demonstrate immunoglobulin produced and released into the culture medium by the stimulated cells were negative. The findings may indicate that among the lymphoid cells responding to the glial antigens under these conditions, there are suppressor cells that abrogate the killer cell effect.     

Antibody-induced redistribution of normal and tumor associated surface antigens

Redistribution (capping) of normal and tumor-associated surface antigens was studied on murine and human cells by the indirect membrane immunofluorescence (MIF) technique. The capping of H-2 isoantigens was compared on normal mouse T-lymphocytes and on YAC cells, a Moloney leukemia virus (MLV) induced lymphoma. H-2 and Moloney virus induced cell surface antigen (MCSA) capping was compared on three YAC lines with different MCSA concentrations. H-2 and tumor-associated surface antigen capping was compared on two polyoma induced sarcoma lines and five methylcholanthrene induced sarcoma lines. In the human system, IgM-capping was compared on normal lymphocytes and on the Burkitt lymphoma derived Daudi line. Capping of HL-A and the Epstein-Barr virus (EBV) determined membrane antigen (MA) was compared on the Burkitt lymphoma derived line Maku and on EBV-superinfected Daudi cells. H-2 antigens on normal murine cells capped more promptly and on a larger fraction of the cell population on the various tumor cells. Surface associated IgM showed a better capping on normal lymphocytes than on Daudi cells. All tumor associated antigens except MCSA, showed good capping. MCSA was almost completely refractory to capping. Increasing concentrations of MCSA appeared to inhibit the capping of H-2 on the YAC sublines with different concentrations of MCSA. The polyoma induced ascites sarcoma (SEWA) did not cap either with regard to H-2 or the polyoma determined surface antigen.